Chlamydia trachomatis is among the most significant bacterial pathogens afflicting humans. Despite this enormous impact on public health, many aspects about C. trachomatis including basic biology, genetics, and pathogenesis are poorly understood. This is reflective of a primary limitation in chlamydial research: the lack of a system for directed genetic manipulation. Two significant achievements were recently reported: a method for introducing DNA into Chlamydia (transformation) and development of a plasmid stable in both E. coli and Chlamydia with an effective selectable antibiotic marker (shuttle vector). This method and tool can enable us, for the first time in the Chlamydia field, the ability to directly introduce desired DNA sequences into Chlamydia and evaluate resulting biological effects, define virulence components, and better understand how disease is caused and ameliorated. Tightly controlled gene expression and targeted gene repression is essential to appropriately analyze the biological effect of a given protein in Chlamydia, especially given the many stages of infection and development. Many molecular components to achieve controlled gene expression and repression have been employed successfully in model bacterial systems; however, none have been tested in Chlamydia. Three specific aims are proposed; 1) Conditional gene expression, 2) Targeted gene repression, and 3) Fluorescent protein validation. Developing these molecular tools in Chlamydia would be pivotal accomplishments in the chlamydial field. Moreover, would allow for the first time the ability to directly demonstrate and define mechanisms for chlamydial pathogenesis.